Smilagenin and its use

ABSTRACT

The invention discloses the use of a smilagenin in the treatment of cognitive dysfunction and similar conditions. Methods of treatment, and pharmaceutical compositions are also disclosed.

CROSS-REFERENCE To RELATED APPLICATION

[0001] This application is a divisional application of copending U.S.patent application Ser. No. 09/362,328, filed Jul. 28, 1999.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to smilagenin and its use intreating cognitive dysfunction and allied conditions; and tocompositions for use in such treatments. The invention is also concernedwith the treatment of conditions that are characterized by a deficiencyin the number or function of membrane-bound receptors. In the following,the present invention will be described principally with reference tothe treatment of Alzheimer's disease (AD) and senile dementia of theAlzheimer's type (SDAT), where deficiencies in a number of receptortypes have been demonstrated. However, it is to be understood that thepresent invention relates generally to the treatment of conditionsattributable to intrinsic pathological conditions and/or exposure toadverse environmental conditions these conditions being characterized bya deficiency in the number or function of membrane-bound receptors or adeficiency in transmission at the junctions between neurons or at thejunctions of neurons and effector cells.

[0003] Conditions of the type mentioned above include Parkinson'sdisease, Lewi body dementia, postural hypertension, autism, chronicfatigue syndrome, Myasthenia Gravis, Lambert Eaton disease, diseases andproblems associated with Gulf War Syndrome, occupational exposure toorganophosphorus compounds and problems associated with aging.

[0004] Alzheimer's disease (AD) and senile dementia of the Alzheimer'stype (SDAT) are grave and growing problems in all societies where,because of an increase in life expectancy and control of adventitiousdisease, the demographic profile is increasingly extending towards amore aged population. Agents which can treat, or help in the managementof, AD/SDAT are urgently required.

[0005] Age-associated memory impairment (MMI) is a characteristic ofolder patients who, while being psychologically and physically normal,complain of memory loss. It is a poorly defined syndrome, but agentswhich are effective in treatment of AD/SDAT may also be of value inthese patients.

[0006] Research into AD/SDAT is being carried out by traditional andconventional medical research methods and disciplines. In conventionalmedicine, there are several approaches to the treatment of AD/SDAT. Itis known that the biochemical processes subserving memory in thecerebral cortex are (at least in part) cholinergically-mediated. Thoseskilled in the art will know that “cholinergically mediated” mechanismsmay be directly attributable to acetylcholine acting on receptors, andthese are direct effects. Other, clinically useful effects may also becaused by modulation of release of acetylcholine from pre-synaptic nerveendings or inhibition of enzymes that destroy acetylcholine. Thesemodulating factors may be exerted through neurones where the mediator isnon-cholinergic; these are referred to as indirect effects. Someattempts at treatment have focussed on the role of other mediators suchas 5-hydroxytryptamine, which is a mediator in other areas of brain,such as the mid-brain nuclei. However, since fibres from these areas areprojected forward into the cerebral cortex where the primary transmitteris acetylcholine, attention has focussed on the management of thismediator in the search for appropriate therapeutic agents.

[0007] Cholinergic strategies for the treatment of AD/SDAT have beendirected at several points along the pathway of formation, synapticrelease and removal of released acetylcholine.

[0008] One approach involves treatment with high doses of lecithin andother precursors of acetylcholine. This is of limited use in producingsustained improvements in cognitive performance.

[0009] Another approach involves the use of vegetable drugs such asPolygalae root extract, which has been shown to enhancecholine-acetylcholine transferase (CAT) activity and nerve growth factor(NGF) secretion in brain. Oral administration of NGF has no effect oncentral nervous system neurons because it is a high molecular weightprotein that cannot pass through the blood-brain barrier. However,agents which can pass through the blood-brain barrier and have astimulating effect on NGF synthesis in the central nervous system havebeen proposed for the improvement of memory-related behavior.

[0010] The results of a third clinical approach, which usescholinesterase inhibitors such as tacrine hydrochloride, have beenmarginally more positive than the above. Substances obtained from plantsused in Chinese and Western medicine, for example huperzine,galanthamine, and physostigmine have all been shown to be ofsome—although limited—benefit in the treatment of AD/SDAT in clinicalstudies and also in laboratory models. All of these substances areinhibitors of acetylcholine esterase (AChE). In patients with AD/SDAT,there may be reduced synthesis of acetylcholine (ACh), reducedefficiency in release of ACh from presynaptic stores, and a decrease inthe number or function of postsynaptic (M₃) receptors. Reductions inpre-synaptic M₂ receptors have also been shown. The beneficial effect ofAChE inhibitors is attributed to enhancement of acetylcholine levels atsynapses in brain by slowing down the destruction of releasedtransmitter.

[0011] Compositions which modulate cholinergic function are known toaffect memory and recall. For example, nicotine stimulates nicotinicacetylcholine receptors, and the short lived memory enhancing effects ofcigarette smoking are thought to be due to the effect of nicotine.Scopolamine, an antagonist of acetylcholine, will produce amnesia andimpaired cognitive function manifesting in psychomotor tests as aprolongation of simple reaction times, possibly as a result of impairedattention, and is used for this purpose as an adjunctive analgesictreatment. The amnesic effect of scopolamine can be antagonized bynicotine.

[0012] There are two families of nicotinic receptor subtypes (a and P),and each includes four subgroups which differ in ligand specificity. Therole of nicotinic receptors in the CNS is not well understood at themolecular level. It is possible that agents binding to nicotinicreceptors may modify the rate of turnover at muscarinic receptor sitesin brain. Nicotinic receptors are ligand-gated ion channels, and theiractivation causes a rapid (millisecond) increase in cellularpermeability to Na⁺ and Ca⁺⁺, depolarisation and excitation.

[0013] Another class of cholinergic receptors can be stimulated bymuscarine. Such muscarinic (M) receptors are G protein-coupledreceptors. Responses of muscarinic receptors are slower; they may beexcitatory or inhibitory. They are not necessarily linked to changes inion permeability. Five types of muscarinic receptors have been detectedby cholinergic receptor cloning, and are designated as m₁-m₅.Pharmacological effects are associated with four of the cloned receptorsand they are designated as M₁-M₄ based on pharmacological specificity.

[0014] Using specific receptor proteins and monoclonal antibodies, ithas been possible to further localize muscarinic receptors in brain asm₁ (postsynaptic) and m₂ (presynaptic). In heart, M₂ receptors arepostsynaptic. Presynaptic muscarinic receptors are thought to beinhibitory, the binding of ACh to these receptors attenuating therelease of further ACh to provide a negative feedback mechanism for AChrelease. Selective M₂ receptor antagonists which are preferentiallydistributed to the brain may therefore be useful in treating Alzheimer'sdisease.

[0015] It is known that, in disease states such as AD/SDAT, there isgeneral neuronal loss and deficits in cholinergic nerve function. It hasbeen speculated that the high affinity nicotinic binding sites in theremaining cholinergic neurons might be converted to low affinity bindingsites in treating such diseases, thereby sustaining transmitter release.By lowering the affinity of the nicotinic binding sites, a quickdesensitising process is avoided.

[0016] Agonist activation at nicotinic receptors in brain has rapidonset and offset. A decreased affinity of the nicotinic receptors willreduce the desensitisation process. Schwarz R. D. et al (J. Neuro Chem42, (1984), 1495-8) have shown that nicotine binding sites arepresynaptically located on cholinergic (and also 5-hydroxytryptaminergicand catecholaminergic) axon terminals. A change in high affinity bindingsites on AD/SDAT may also induce a change in the modulatory effect thenicotinic binding sites may have on other transmitter systems.

[0017] Presynaptic cholinergic mechanisms are also under inhibitorycontrol by GABAergic neurons and this inhibition is thought to beintensified in AD/SDAT. Removal or reduction of this inhibitionintensifies presynaptic cortical cholinergic activity and enhancescognitive processing.

[0018] The interactions of interneuronal fibres innervated by nicotine(reducing binding affinity), and dis-inhibition of GABAergic fibres bothhave a presynaptic locus.

[0019] This is a simplistic model of central transmission, but providesa framework for understanding the attempts which have been made toincrease the effective concentration of acetylcholine in centralsynapses. This further illustrates thc concept of direct and indirectaction. There are disadvantages attaching to the three conventionaltherapeutic approaches to AD/SDAT treatment mentioned above: AChprecursor supplementation, agonist replacement and acetylcholineesterase inhibition. These treatments may result in a short-termincrease in the availability of ACh which may activate feedbackmechanisms resulting in the desensitisation of postsynaptic receptors.On theoretical grounds, long term benefits would not be predicted andwhen treatment is interrupted, any benefits in management of AD/SDAT andAAMI disappear and the condition may even be aggravated.

[0020] It has been shown that a compound with M₁ agonist and M₂/M₃antagonist activity improved cognitive performance in SDAT patients(Sramak et al, Life Sciences vol. 2, No. 3,195-202,1997). However, thiscompound causes unacceptable cholinergic side effects, such as fatigue,diarrhea and nausea.

[0021] A more radical approach to AD/SDAT and AAMI aims to increase thenumber of postsynaptic (M₁) receptors, in brain. It is known fromChinese Patent No. CN1096031A, that sarsasapogenin (SaG) can up-regulateM₁ cholinergic receptors and also down-regulate (i.e. move towardsnormal levels of) β-adrenergic receptors, the number of which may bepathologically-raised in AD/SDAT.

[0022] Patent applications have been published which claim theusefulness of a number of steroid sapogenins having spirostane,furo-spirostane, spirosolane or solanidine structures in the treatmentof diseases including SDAT. Two patent publications are of particularrelevance here: Chinese patent publication No CN1096031A claims the useof the spirostane sapogenin, sarsasapogenin, in the treatment of SDAT.The disclosure in this document, however, is brief. The other documentof relevance is patent publication DE 4303214A1 which claims the use ofa very wide range of saponins and sapogenins in the treatment of a wholerange of diseases that the inventors consider to be of viral origin.This disclosure is however of dubious value in that it is wellrecognized that there is no infective element to a very large number ofthe conditions that are characterized by deficient synaptic transmissionand thus the basic premise of the alleged invention is flawed. Inaddition they present no data of any kind that allows one skilled in theart to be able select a preferred compound from the large number thatare claimed.

[0023] The inventors have found that smilagenin (SMI) exhibits theability to regulate receptors. In particular, this compound has beenfound to increase the number of M2 receptors in the brain. Thus,according to one aspect of the invention, there is provided the use ofsmilagenin in the manufacture of a medicament for the treatment of acondition characterized by a deficiency in postsynaptic membrane-boundreceptor number or function.

[0024] Those skilled in the art will be aware of the relationshipbetween saponins and their sapogenins, and that the latter tend to befat-soluble whereas the saponins tend to be water-soluble. Sapogeninsare therefore better able to cross the blood-brain barrier. The skilledman will also be aware of the epimerisation of certain sapogenins underconditions of acid hydrolysis.

[0025] The sapogenin of interest in this invention has the followingformula:

[0026] With reference to this general formula, smilagenin has the A/Bring conformation as cis and the stereochernical configuration at theC25 methyl group is R. The hydroxyl group on the spirostane ring is inthe 3β-OH position.

[0027] Smilagenin occurs naturally in a range of plant species, notablyfrom the genera Smilax, Asparagus, Anemarrhena, Yucca and Agave. Thespecies presently of greatest interest include Smilax regelii Kilip &Morton—commonly known as Honduran sarsaparilla, Smilaxaristolochiaefolia Miller—commonly known as Mexican sarsaparilla; Smilaxornata Hooker—commonly known as Jamaican sarsaparilla; Smilaxaspera—commonly known as Spanish sarsaparilla; Smilax glabra Roxburgh;Smilax febrifuga—Kunth—commonly known as Ecuadorian or Peruviansarsaparilla, Anemarrhena asphodeloides Bunge; Yucca schidigera Roezi exOrtgies; and Yucca brevifolia Engelm.

[0028] According to a further aspect of the present invention, there isprovided a pharmaceutical composition having cognitive functionenhancing properties which comprises an effective amount of smilagenin.

[0029] In another aspect, the invention provides a pharmaceuticalcomposition having cognitive function enhancing properties whichcomprises an effective amount of smilagenin in the form of an extractderived from a plant of the genus Smilax, Asparagus, Anemarrhena, Yuccaor Agave.

[0030] It will be appreciated that the invention embraces within itsscope the use of the compositions defined above. Thus, according to afifth aspect, the present invention provides a method of enhancingcognitive function which comprises administering to a human or animal aneffective dosage of a composition of the invention.

[0031] The invention also provides a method of enhancing cognitivefunction in a human or non-human animal, which comprises administeringan effective dose of smilagenin.

[0032] As used herein, the term “cognitive function” refers to functionssuch as thinking, reasoning, remembering, imagining and learning.

[0033] In identifying compounds that would have use in the treatment ofSDAT and other diseases characterized by reductions in receptor numbersor synaptic transmission, the inventors have given consideration to theneed to identify compounds that would have the desired effect but wouldbe devoid of any oestrogenic effects, as these would be unacceptable,particularly in male patients. A number of the compounds claimed to haveactivity in patent application DE 4303214A1 have marked oestrogenicactivity and are therefore unacceptable. Smilagenin, however, does notdisplay oestrogenic activity. In addition this compound was tested atother steroid receptors and was found to have no activity at any of thefollowing receptors:

[0034] Progesterone

[0035] Glucocorticoid

[0036] Testosterone

[0037] Smilagenin has also been tested for activity in a number ofin-vitro assays. The assays/experiments that were considered of keyimportance in determining possible activity in the elevation of membranebound receptor numbers were as follows:

[0038] 1. Chinese hamster ovary (CHO) cells transfected with the a DNAfragment coding for a muscarinic receptor. The cell line used for themajority of the experiments was a cell line expressing the m2 receptor.

[0039] 2. The effects of muscarinic receptor expression in cultured celllines of neuronal origin were investigated.

[0040] 3. Cultured cardiac muscle cells obtained from neonatal SpragueDawley rats. The cardiac muscle cells express muscarinic receptors,typically m2. The level of these receptors fails on prolonged cultureand the effects of compounds of interest in preventing the fall inreceptor numbers was investigated.

[0041] The methods and the results of these experiments are nowdescribed in turn.

[0042] 1 CHO Cell Line Experiments

[0043] The effects of various compounds on the expression of m2receptors on CHO cells transfected with DNA for the m2 receptor wereinvestigated. Receptor numbers were assayed using tritiated QNB bindingand subtracting non-specific binding. Compounds were dissolved in DMSOand DMSO was used as a control. Compounds were tested at a range offinal concentrations. Compounds were also tested in the presence andabsence of tamoxifen to try to distinguish an oestrogen receptormediated mechanism. The results are summarized in the Table 2 below,where the compound used in the invention appears in bold, and data onother sapogenins is given for comparative purposes: TABLE 2 Effects ofsmilagenin on the expression of m₂ receptors on CHO cells Effect onreceptor expression - given as % increase compared to Molarconcentration of control (negative values in Compound compound brackets)Sarsasapogenin 10⁻⁵ 34 10⁻⁶ (14) Anzurogenin D 10⁻⁵ 22 10⁻⁶ (26)Sisalgenin 10⁻⁵ NS 10⁻⁶ NS Smilagenin 10⁻⁵ 57 10⁻⁶ 18 Diosgenin 10⁻⁵ NS10⁻⁶ NS Ruscogenin 10⁻⁵ (22) 10⁻⁶ NS Tigogenin 10⁻⁵ NS 10⁻⁶ NS

[0044] Thus the experiments indicate that smilagenin was able toincrease the number of muscarinic receptors expressed on the surface ofCHO cells cultured in-vitro. The effect was not antagonized bytamoxifen, indicating that the mechanism involved did not involve theoestrogen receptor.

[0045] Other in vitro assays have been employed to establish the effectsof smilagenin. In particular various neuroblastoma cell lines includingSKN-SN and SH-SY5Y cells as well as phaechromoacytoma cell lines havebeen cultured in vitro in the presence of β-amyloid fragments or serumdepletion. A number of techniques to demonstrate the effectiveness ofthe compounds in protecting the cultured cells were investigated. Thesetechniques included Trypan blue exclusion, chemiluminescence and releaseof lactate dehydrogenase. Of most interest was the observation thatincubation of cells, in particular PC12 cells, with β-amyloid reducedthe number of muscarinic receptors measured using radio-labeled ligandbinding techniques. This reduction in receptor numbers was found to beameliorated by smilagenin.

[0046] 3 Effects of Smilagenin on Cultured Cardiac Muscle Cells.

[0047] Cardiac muscle cells were isolated from the ventricular muscle ofneonatal Sprague Dawley rats using standard techniques. Cells werecultured in vitro and muscarinic receptor numbers expressed on cellsurfaces membrane fragments after homogenization of cells harvested atvarious time points were estimated using specific binding of tritiatedQNB. Preliminary experiments demonstrated that the number of receptorsexpressed tended to decline after 10 days of culture. The experimentswere therefore designed to investigate the effects of the variouscompounds in inhibiting this decline in receptor numbers.

[0048] The results of these experiments are summarized in Table 3, wherethe compound used in the invention appears in bold, and data on othersapogenins is given for comparative purposes: TABLE 3 Effects of variouscompounds on muscarinic receptor expression on cultured cardiac musclecells Concentration of compound causing a significant increase in numberof receptors expressed on neonatal cardiac muscle after 10 days in vitroCompound culture Diosgenin NS Anzurogenin D 10⁻⁶M Ruscogenin NSSarsasapogenin 10⁻⁵M Tigogenin NS Astragaloside 10⁻⁵M Smilagenin 10⁻⁶M

[0049] It is speculated here that the effect of the active compoundclaimed in this patent may operate through an effect on G protein andthat the effects on receptor numbers are secondary to an effect onG-protein. When a membrane bound G-protein linked receptor is stimulatedtwo basic sets of events are initiated: the effecter response; and theinternalization of the receptor. The subsequent processing of thereceptor to the state where it is again in a form on the cell surface orother membrane surface where it can interact with another receptorligand appears to be subject to a number of factors. A number of thesefactors or mechanisms appear to be G-protein linked. There is evidencethat activation of m₃ receptors may have an effect on G-proteinexpression or levels. It is speculated that the actions of the compoundsdescribed in this patent may due to an interaction in the processes ofreceptor regeneration, G-protein linkage or G-protein homeostasis.

[0050] An alternative hypothesis is that the compounds are increasingthe synthesis or release or a decreased rate of degradation ofneurotropic factors such as brain derived growth factor and/or nervegrowth factor. These effects on growth factors might be due to an effectof the compound on a cytosolic or nuclear receptor or the binding of acompound to a promoter region with a consequent effect directly on therate of production of mRNA for the growth factor or as a consequence ofincreasing the production of another material factor such as G-proteinor finally the effects may be secondary to an effect on receptor orG-protein procession.

[0051] The increased expression and/or abnormal processing of theamyloid precursor protein (APP) is associated with the formation ofamyloid plaques and cerebrovascular amyloid deposits which are the majormorphological hallmarks of Alzheimer's disease. Of particular interestare the processes regulating the proteolytic cleavage of APP intoamyloidogenic and nonamyloidogenic fragments. The cleavage of APP by theenzyme α-secretase within the β-amyloid sequence of the protein resultsin the formation of a non amyloidogenic C-Terminal fragment, and thesoluble APPsα fragment; this latter fragment has been shown to haveneurotropic and neuroprotective activity as well as to enhance memory inmice when injected intra-cerebro-ventrically (ICV). In contrast,processing of APP by β-secretase exposes the N-terminus ofβ-amyloid.which is released by γ-secretase cleavage at the variableC-terminus. The resulting β-amyloid peptides, which contain 39-43 aminoacids, have been shown to be neurotoxic and to accumulate in plaqueswhich interfere with inter-neurone connections.

[0052] A number of studies have shown that stimulation of theprotein-kinase (PKC) linked muscarinic M₁ and M₃ receptors results in anincrease in a-secretase activity. As a consequence processing of APP toAPPsα with its neuroprotective effects is increased. In parallel,processing of APP by β- and γ-secretase is decreased and there is aconsequential reduction of β-amyloid. Other transmitters such as nervegrowth factor (NGF) and brain derived neurotropic factor (BDNF) as wellas bradykinin and vasopressin may have similar effects in increasing theproportion of APP processed to APPsα. There may be a number of factorsinvolved in the effects of NGF which may include binding of the factorto the tyrosine kinase receptor (TrkA) and the stimulation ofphospholipase Cγ with subsequent phosphorylation and activation ofprotein kinase C (PKC) and increase in relative activity of α-secretase.

[0053] Any treatment which increases activity of protein-kinase Cselectively in brain might therefore be expected to be of use in themanagement of Alzheimer's disease. Until recently agonists selective atthe M₁ receptor have not been available. Non-selective agonists would beexpected to stimulate pre-synaptic M₂ receptors which cause negativefeedback and hence would further severely impair muscarinictransmission. Selective agonists at the M₁ receptor are now becomingavailable (talsaclidine) and such agents are under investigation for thetreatment of AD. There is however, a substantial risk that, as with thechronic administration of any receptor agonist, the clinical benefitsseen will be severely limited in terms of the size of benefit byreducing receptor numbers or reducing sensitivity and in terms of sideeffects due to lack of receptor specificity. Thus compounds as describedin this invention, which selectively increase muscarinic M₁ receptornumbers, with little or no effect on muscarinic M₂ receptor numbers inthe brain would be expected to be devoid of the problems seen with amuscarinic agonist and hence have particular utility. Indeed thebenefits may be seen in three parts as follows.

[0054] 1. A selective increase in M₁ receptor numbers leading toincreased synaptic transmission. Chronic administration of a selectiveagonist will, at best, have no adverse effect on transmission;

[0055] 2. Secondary to the increased receptor numbers, an increasestimulation of PKC with a consequential increase in α-secretaseactivity, leading to:

[0056] 2.1 A reduced production of β-amyloid and a consequent reductionof plaque formation and neuronal loss;

[0057] 2.2 An increase in APPsα and a consequent improvement in cerebralfunction as witnessed by an improvement in short and long term memory.

[0058] In order to illustrate the invention further by way ofnon-limiting example, reference will now be made to the accompanyingdrawings and to the Examples which follow; in the drawings:

[0059]FIG. 1 illustrates a hypothetical mode of action for smilagenin;

[0060]FIG. 2 illustrates the results obtained in Example 1 below;

[0061]FIG. 3 illustrates the results obtained in Example 2 below; and

[0062]FIG. 4 illustrates the results obtained in Example 3 below.

[0063] Smilagenin is represented in the drawings by the abbreviationSMI.

[0064] Referring to FIG. 1, a diagrammatic representation of thefunction of smilagenin is shown. It is believed that smilagenin actsprimarily on cell nuclei; the invention is not, however, limited to anyparticular mode of action. The observed increase in muscarinic receptornumber consequential upon administration of smilagenin is interpreted asleading to increased expression of muscarinic receptor protein. Thepossible link between the secretases and β-amyloid protein formation(discussed above) is indicated in the drawing.

[0065] The following Examples are provided to illustrate the inventionin a non-limiting manner.

EXAMPLE 1

[0066] In a CHO cell line expressing recombinant human muscarinicreceptors in vitro, the number of muscarinic receptors tends to declinewith time. Smilagenin (1-10 μM) incubated for 72 hours increasesmuscarinic receptor density.

[0067] Methods:

[0068] Effect of smilagenin on muscarinic receptor density in CHO cellsexpressing recombinant human muscarinic receptors.

[0069] Chinese hamster ovary (CHO) cells expressing high levels ofreceptor (˜2.2 pmoles receptor/mg protein) were cultured in flasks (150ml) for 24 hours before the start of the experiment. Vehicle (DMSO) andsmilagenin (at 1 and 1 0μM) were added to the medium for 48 h. Theculture medium was discarded, the cells scraped off and resuspended inHanks solution, centrifuged and m-receptor levels determined byincubating with [³H]-QNB for 30 min followed by liquid scintillationcounting. Protein levels were determined by a micro Lowry method.

[0070] Results:

[0071] These are illustrated in FIG. 2. Over the culturing periodtreatment with smilagenin prevents the decrease in muscarinic receptornumber in a concentration-dependent manner.

EXAMPLE 2

[0072] Smilagenin (18 mg/kg/day) administered in the food over 3 monthsreversed the decline in muscarinic (M) receptor number in the brain overthis period, restoring levels to close to those observed in youngcontrol animals.

[0073] Methods:

[0074] Single point analysis of brain M receptor density.

[0075] Sprague-Dawley rats, 23-month-old, (i.e. aged) were divided into2 groups, aged-control and aged+smilagenin. Smilagenin (18 mg/kg/day)was mixed in the chow over the 3 month period; Male 4-6 month old ratsacted as young controls. At the end of the 3-month treatment period,pairs of treated and control animals were sacrificed by cervicaldislocation and the intact brain removed. Single point analysis of brainM receptor density was obtained.

[0076] Results:

[0077] Compared to the young controls there were reductions in Mreceptor density in brain of the aged controls (FIG. 3). Compared toaged controls, smilagenin increased M receptor numbers. Groups treatedwith smilagenin were significantly different from untreated controls(FIG. 3).

EXAMPLE 3

[0078] Smilagenin (18 mg/kg/day) administered in the food over 4 monthsreversed the decline in cognitive function over this period compared tothat observed in young control animals (FIG. 4).

[0079] Methods:

[0080] Cognitive function in the Y-maze test

[0081] Sprague-Dawley rats aged 23 months (i.e. aged) were divided intoaged-control or aged+smilagenin treatment group. Rats aged 3 to 4 monthsacted as young controls. The treatment group received 18 mg/kg/day ofsmilagenin in chow. Chow without smilagenin was given to each rat afterthe chow containing smilagenin had been consumed. Three months later theanimals were tested in the Y-maze as follows; an equilateral Y-shapedmaze was used composed of three 45 cm arms with copper rods in the floorat the end of each arm which conduct electricity and which were 0.2 cmin diameter, 14 cm in length and with a 1 cm gap between them. Therewere 15 W signal lights at each end. A safe region was indicated by alight signal in an arm where there was no electricity. If a rat moved toa safe region, a correct reaction was recorded. If it moved to an armwhere there was no light, an error reaction was recorded. Following theresponse, an electric shock was again given after a 5 seconds interval.Twenty trials in total were conducted and the correct reaction rate wasobtained: number of correct reactions/(20)/mean response time. Sevensuch training sessions were completed over seven days. Memory tests werethen repeated 15 and 30 days later (=4 months) using the same techniquesas just described.

[0082] Results:

[0083] Compared to young control rats, aged control animals have areduced learning and memory performance in the Y-maze tests. A regime of3 months intake of smilagenin (18 mg/kg/day) reversed the decline incognitive function (FIG. 4), giving results comparable to those of younganimals.

1. A use of smilagenin in the manufacture of a medicament for thetreatment of a condition characterized by a deficiency in receptornumber, function or turnover.
 2. A method of treating a conditioncharacterized by a deficiency in receptor number, function or turnover,which comprises administering an effective dose of smilagenin.
 3. Amethod as claimed in claim 2 , said method comprising: providing acomposition having receptor number or turnover enhancing properties,said composition including smilagenin; and administering saidcomposition to an organism for treating a condition characterized bysaid deficiency.
 4. The method of claim 2 , wherein said conditionfurther comprises a reduced amount of receptors.
 5. The method of claim2 , wherein said composition further comprises a carrier.
 6. The methodof claim 2 , wherein said condition is found in postsynaptic neurons. 7.The method of claim 2 , wherein said smilagenin is derived fromindividual plants belonging to the genera of Smilax, Asparagus,Anemarrhena, Yucca and Agave, solely or in combinations thereof.
 8. Themethod of claim 2 which comprises administering a foodstuff or beveragehaving an effective amount of smilagenin.
 9. The method of claim 2 whichcomprises administering smilagenin in the form of a pharmaceuticalcomposition.
 10. A use of smilagenin in the manufacture of a medicamentfor the treatment of a condition characterized by the presence ofneurofibrillary tangles and/or β-amyloid plaques.
 11. A method oftreating a condition characterized by the presence of neurofibrillarytangles and/or β-amyloid plaques, which comprises administering aneffective dose of smilagenin.
 12. A method as claimed in claim 11 , saidmethod comprising: providing a composition, said composition includingsmilagenin; and administering said composition to an organism fortreating a condition characterized by said presence of neurofibrillarytangles and/or β-amyloid plaques.
 13. The method of claim 12 , whereinsaid composition further comprises a carrier.
 14. The method of claim 11, wherein said smilagenin is derived from individual plants belonging tothe genera of Smilax, Asparagus, Anemarrhena, Yucca and Agave, sojely orin combinations thereof.
 15. The method of claim 11 , which comprisesadministering a foodstuff or beverage containing an effective amount ofsmilagenin.
 16. The method of claim 11 , which comprises administeringsmilagenin in the form of a pharmaceutical composition.
 17. Apharmaceutical composition having cognitive function enhancingproperties, said composition comprising a pharmacologically effectiveamount of smilagenin.
 18. The pharmaceutical composition of claim 17 ,further comprising a carrier.
 19. The pharmaceutical composition ofclaim 17 , wherein said smilagenin is in the form of an extract derivedfrom a plant of the genus Smilax, Asparagus, Anemarrhena, Yucca andAgave, solely or in combinations thereof.
 20. The pharmaceuticalcomposition of claim 17 , wherein a pharmacologically effective amountof said composition is administered to an organism to treat a condition.21. The pharmaceutical composition of claim 20 , wherein said conditionis characterized by cognitive dysfunction and allied conditions.
 22. Thepharmaceutical composition of claim 21 wherein said condition isAlzheimer's disease, senile dementia of the Alzheimer's type,Parkinson's disease, Lewi body dementia, postural hypotension, autism,chronic fatigue syndrome, Myasthenia Gravis, Lambert Eaton disease,diseases and problems associated with Gulf War Syndrome, occupationalexposure to organophosphorus compounds and problems associated withaging.
 23. The pharmaceutical composition of claim 20 , wherein asymptom of said condition includes a deficiency of receptors.
 24. Thepharmaceutical composition of claim 23 , wherein said deficiencycomprises a reduction in receptor number, function or turnover, orcombination thereof.
 25. The pharmaceutical composition of claim 20 ,wherein said condition is characterized by a presence of neurofibrillarytangles and/or β-amyloid plaques.
 26. A foodstuff or beverage comprisingan effective dosage of smilagenin.
 27. The foodstuff or beverage ofclaim 26 , wherein said smilagenin is in the form of an extract derivedfrom a plant of the genus Smilax, Asparagus, Anemarrhena, Yucca andAgave, solely or in combinations thereof.
 28. The foodstuff or beverageof claim 26 , wherein an effective amount of said composition isadministered to an organism to treat a condition.
 29. The foodstuff orbeverage of claim 28 , wherein said condition is characterized bycognitive dysfunction and allied conditions.
 30. The foodstuff orbeverage of claim 29 , wherein said condition is Alzheimer's disease,senile dementia of the Alzheimer's type, Parkinson's disease, Lewi bodydementia, postural hypotension, autism, chronic fatigue syndrome,Myasthenia Gravis, Lambert Eaton disease, diseases and problemsassociated with Gulf War Syndrome, occupational exposure toorganophosphorus compounds and problems associated with aging.
 31. Thefoodstuff or beverage of claim 28 , wherein a symptom of said conditionincludes a deficiency of receptors.
 32. The foodstuff or beverage ofclaim 31 , wherein said deficiency comprises a reduction in receptornumber, function or turnover, or combination thereof.
 33. The foodstuffor beverage of claim 28 , wherein said condition is characterized by thepresence of neurofibrillary tangles and/or β-amyloid plaques.
 34. Amethod of testing a compound for efficacy in improving the functionand/or increasing and/or stabilizing a population of a specific receptoror type of receptor, the method comprising: preparing or retrievingsuitable cells transfected with DNA for a specific receptor or type ofinterest; dividing the transfected cells into separate portions, a firstportion of said separate portions to serve as a control sample, and asecond portion of said separate portions to serve as a test sample;allowing said control sample and test sample to grow in the presence ofa nutrient medium; removing the nutrient medium from said control sampleand test sample and then (a) adding the compound dissolved in acytologically acceptable carrier to the test sample and (b) adding anequivalent amount of said carrier to the control samples; and performingan assay or assays to determine the function, number or turnover of saidspecific receptor or type of receptor present in the control sample andtest sample.
 35. The method of claim 34 , wherein said suitable cellsfor transfection are Chinese Hamster Ovary cells.
 36. The method ofclaim 34 , wherein said specific receptor is a muscarinic receptor. 37.The method of claim 34 , wherein said compound comprises smilagenin. 38.A method of testing a compound for efficacy in reducing a decline innumber, over time, of specific receptor or type of receptor in acultured cell line, the method comprising: culturing, in a culturemedium, a population of cells expressing a specific receptor or type ofreceptor; dividing said population of cells into a control portion and atest portion; culturing said control portion and test portions underapproximately similar conditions, wherein said test portion of cells isexposed to a compound; performing assays to determine the decline of thespecific receptor or type of receptor of interest in said controlportion of cells and in said test portion of cells exposed to saidcompound over a time period; and comparing, at a predetermined time, theamounts of said specific receptor or type of receptor of interest insaid control portion and test portions of cells.
 39. The method of claim38 , further comprising determining an effect of said compound on thedecline of said specific receptor or type of receptor of interest. 40.The method of claim 38 , wherein said compound is comprised ofsmilagenin.